DE10021688A1 - New DNA sequences involved in isoprenoid biosynthesis, useful in screening for compounds with e.g. antimicrobial and herbicidal activity - Google Patents

Abstract

DNA sequence (I) that encodes one of sequences (5) or (14), of 539 and 343 amino acids (aa), respectively, or their analogs or derivatives in which one or more aa are deleted, inserted or exchanged, provided that enzymatic activity of the protein is not significantly reduced. Independent claims are also included for the following: (1) DNA sequence (Ia) comprising (I) plus a promoter and 3'-untranslated sequences that are functional in viruses, eukaryotes or prokaryotes; (2) expression vector containing at least one (I); (3) protein (II) that is encoded by (i) sequences (1; 1920 bp) or (9; 1320 bp), (ii) sequences that hybridize to (1) or (9), or to their fragments that encode mature proteins, or (iii) sequences that would, but for the degeneracy of the genetic code, hybridize with (ii); (4) plant cells containing (I); (5) transformed plant cells, and transgenic plants regenerated from them, containing (I); (6) transgenic viruses, eukaryotes and prokaryotes containing (I) and able to produce isoprenoids; (7) method (M1) for isolating (II) from the culture supernatant or lysate of parasites, by chromatographic and electrophoretic techniques; (8) method (M2) for isolating (II) by expression of exogenous DNA; (9) method (M3) for determining the enzymatic activity of LytB and YfgB proteins; (10) method (M4) for preparing organisms of (6); and (11) method (M5) of screening for compounds (A) for antimycotic, antibiotic, antiparasitic, antiviral, bactericidal, fungicidal and herbicidal activities.

Description

The present invention relates to DNA sequences which at Inte in the genome of viruses, eukaryotes and prokaryotes change isoprenoid biosynthesis and genetic engineering Process for the production of these transgenic viruses, eukaryotes and prokaryotes. It also concerns identification procedures decoration of fabrics with herbicidal, antimicrobial, antipa anti-viral, anti-viral, anti-fungal, anti-bacterial effect Plants and antimicrobial, antiparasitic, antifungal shear, antibacterial and antiviral effect in humans and Animal.

The biosynthetic pathway for the formation of isoprenoids via the klas acetate / mevalonate path and an alternative, mevalo nate-independent biosynthetic pathway, the deoxy-D-xylulose Phosphate path (DOXP path) is already known (Rohmer, M., Kna ni, M., Simonin, P., Sutter, B., and Sahm, H. (1993): Biochem. J. 295: 517-524).

However, it is not known how and how in viruses, Eukaryotes and prokaryotes change the isoprenoid cone concentration via the deoxy-D-xylulose-Phoshat route that can.

There are therefore DNA sequences available for Encode enzymes involved in the DOXP pathway. Both genes (lytB and yfgB) and enzymes (LytB and YfgB) are on the isoprene Noid biosynthesis is involved and for the survival of each Organisms essential (Examples 1 and 2).

The invention relates to the following DNA sequences: DNA sequences for a polypeptide with the in SEQ ID NO: 5 encode amino acid sequence shown or for an analog or derivative of the polypeptide according to SEQ ID NO: 5, wherein one or multiple amino acids deleted, added or by others Amino acids have been substituted without the enzymatic Significantly reduce the effect of the polypeptide, and  DNA sequences for a polypeptide with that in SEQ ID NO: 14 encode amino acid sequence shown or for an analog or derivative of the polypeptide according to SEQ ID NO: 14, wherein one or deleted or added several amino acids or by an whose amino acids have been substituted without the enzymati to reduce the effect of the polypeptide significantly.

The invention is further defined by the claims to. Developments of the invention are de by the dependent claims finishes.

The genes and their gene products (polypeptides) are listed in the sequence listing with their primary structure and have the following assignment:
SEQ ID NO: 1: lytB gene
SEQ ID NO: 5: LytB protein
SEQ ID NO: 9: yfgB gene
SEQ ID NO: 14: YfgB protein

The DNA sequences all come from Plasmodium falciparum, strain 3D7.

In addition to the DNA sequences mentioned in the sequence listing are also those suitable due to the degeneration of the genetic Codes have a different DNA sequence, but for the same one Polypeptide or for an analog or derivative of the polypeptide according to, wherein one or more amino acids are deleted adds or has been substituted by other amino acids, without significantly reducing the enzymatic effect of the polypeptide reduce.

The sequences according to the invention are suitable for the overexpress sion of genes in viruses, eukaryotes and prokaryotes which are responsible for the isoprenoid biosynthesis of the 1-deoxy-D-xylulose pathway are literal.  

According to the invention belong to the eukaryotes or eukaryotic Animal cells, plant cells, algae, yeasts, fungi and to the prokaryotes archaebacteria and eubacteria.

When a DNA sequence is integrated into a genome on which one of the DNA sequences given above is located Expression of the genes described above in viruses, eukaryotes and prokaryotes. The transform according to the invention Viruses, eukaryotes and prokaryotes are inherently known as is known and the isoprene formed in the meantime noid isolated and cleaned if necessary. Not all isoprene noides need to be isolated because the isoprenoids in some Cases are released directly into the air.

The invention further relates to a method for producing transgenic viruses, eukaryotes and prokaryotes with isoprenoid Expression that contains the following steps.

• a) Production of a DNA sequence with the following partial sequences
  • a) promoter which is active in viruses, eukaryotes and prokaryotes and ensures the formation of an RNA in the intended target tissue or cells,
  • b) DNA sequence coding for a polypeptide with the amino acid sequence shown in SEQ ID NO: 5 or 14 or for an analog or derivative of the polypeptide according to SEQ ID NO: 5 or 14,
  • c) 3'-untranslated sequence which leads to the addition of poly-A residues to the 3 'end of the RNA in viruses, eukaryotes and prokaryotes,
• b) transfer and incorporation of the DNA sequence into the genome of viruses, prokaryotic or eukaryotic cells with or without Use of a vector (e.g. plasmid, viral DNA).

The intact cells can be removed from the transformed plant cells zen plants are regenerated.

The sequences coding for the proteins with the nucleotides ab can follow Seq ID NO: 1 and Seq ID NO: 9 with one to ensure scripting in certain organs or cells  Promoter provided in the sense orientation (3 'end of the promoter to the 5 'end of the coding sequence) to the Se sequence encoding the protein to be formed is coupled. On the 3 'end of the coding sequence becomes the termination termination signal determining the mRNA synthesis. Around the protein to be expressed in certain subcellular comparisons such as chloroplasts, amyloplasts, mitochondria, vacuo Conducting le, cytosol or intercellular spaces can be between the promoter and the coding sequence another one for egg ne so-called signal sequence or a transit peptide encoding Sequence. The sequence must be in the same reading frame like the coding sequence of the protein. For preparation the introduction of the DNA sequences according to the invention into higher ones Plants have a large number of cloning vectors bar, which be a replication signal for E. coli and a marker contain a selection of transformed cells leaves. Examples of vectors are pBR 322, pUC series, M13mp- Series, pACYC 184, EMBL 3 etc. Depending on the method of introduction desired genes in the plant, he can further DNA sequences to be demanding. For example, for the transformation of Plant cell that uses the Ti or Ri plasmid must have at least Mostly a right boundary, but often the right and the left boundary of the Ti and Ri plasmid T-DNA as a flank rich in the genes to be introduced. The usage of T-DNA for the transformation of plant cells is inten siv examined and sufficient in EP 120516; Hoekama, in: The Binary Plant Vector System, Offset-drukkerij Kanters B.V. Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant Sci. 4,1-46 and An et al. (1985) EMBO J. 4, 277-287 been. Once the introduced DNA is integrated in the genome, so it is usually stable and remains in the offspring of the originally transformed cells. she receives usually a selection marker that transforms the Plant cells resistance to a biocide or an tibiotic, such as kanamycin, G 418, bleomycin, hygromycin or Phosphinotricin and the like. a. mediated. The individually used Marker should therefore select the transformed cells against over cells that lack the inserted DNA.  

There are many techniques for introducing DNA into a plant ken available. These techniques include transformation with the help of agrobacteria, e.g. B. Agrobacterium tumefaciens, the fusion of protoplasts, the microinjection of DNA, the Electroporation, as well as ballistic methods and the virus infection tion. From the transformed plant material can then suitable medium, which antibiotics or biocides for Selek tion, whole plants can be regenerated again. When it comes to injection and electroporation, there are no special features The demands placed on the plasmids. But should out cells transformed in this way regenerate whole plants the presence of a selectable marker gene is necessary agile. The transformed cells grow within the plant zen in the usual way (McCormick et al. (1986) Plant Cell Reports 5, 81-84). The plants can be grown normally and with plants that have the same transformed genetic makeup or other genes have to be crossed. The result from it the individuals have the corresponding phenotypic properties create.

The invention furthermore relates to expression vectors, ent one or more of the DNA sequences of the invention hold. Such expression vectors are obtained by using the DNA sequences according to the invention with suitable functional Provides regulatory signals. Such regulation signals are DNA sequences responsible for expression in for example promoters, operators, enhancers, ribosomal bin and that are recognized by the host organism.

If necessary, other regulatory signals, which at for example, replication or recombination of the recombinant Control DNA in the host organism, part of the expression vector be tors.

Likewise belong those with the DNA sequences according to the invention or Expression vectors transformed host organisms to the opposite state of the invention.  

Be suitable for the expression of the enzymes according to the invention especially those host cells and organisms that are not intrinsic have enzymes of the DOXP pathway. This is true for Archae bacteria, animals, some mushrooms, slime molds and some eubac terie too. Due to the lack of this intrinsic enzyme activity Detection and purification of the recombinant enzyme me much easier. It also only becomes possible with little effort the activity and especially the inhibition the activity of the recombinant enzymes according to the invention various chemicals and pharmaceuticals in crude extracts from the To measure host cells.

The enzymes according to the invention are advantageously expressed usually in eukaryotic cells when posttranslatori modifications and a native folding of the polypeptide chain should be achieved. In addition, depending on the express sion system in the expression of genomic DNA sequences is enough that introns are removed by splicing the DNA and the Enzymes in the polypep characteristic of the parasites tide sequence are produced. Sequences coding for introns can also by recombinant DNA technology from the ex priming DNA sequences eliminated or experimentally inserted be added.

The isolation of the protein can be from the host cell or the Culture supernatant of the host cell according to Ver drive done. It can also be an in vitro reactivation of the Enzymes may be required.

To facilitate purification, the inventive Enzymes or partial sequences of the enzymes as a fusion protein different peptide chains can be expressed. Suitable for this especially oligo-histidine sequences and sequences by the Glutathione-S-transferase, thioredoxin or calmodulin-binding Peptides are derived. Mergers with thioredoxin-derived Sequences are particularly suitable for prokaryotic expression, since this increases the solubility of the recombinant enzymes becomes.  

Furthermore, the enzymes or partial sequences according to the invention zen of the enzymes as a fusion protein with such, the expert be knew, peptide chains are expressed that the recombinant Enzymes in the extracellular environment or in certain Kompar moments of the host cells are transported. This can be so probably the purification, as well as the investigation of the biological the activity of the enzymes.

The expression of the enzymes according to the invention can be prove to be expedient to change individual codons. there is the targeted exchange of bases in the coding region also useful if the codons used in the parasites depart from codon usage in heterologous expressions system to ensure an optimal synthesis of the protein most. In addition, deletions of untranslated 5 'or 3 'sections make sense, for example if several destabili Sequence motifs ATTTA are present in the 3 'region of the DNA. Then these should be given preferential expression in eukaryotes be deleted. Changes of this kind are deletions, ad ditions or exchange of rabbits and also the subject of present invention.

Furthermore, the enzymes according to the invention can be found under standardi conditions by techniques known to those skilled in the art obtained in vitro translation. Suitable systems are rabbit reticulocyte and wheat germ extracts and bacts rienlysate. In vitro mRNA transcribed in Xenopus Oocytes are translated.

Chemical synthesis can produce oligo- and polypeptides be made, whose sequences from the peptide sequence of the inventions enzymes according to the invention are derived. With a suitable choice of Sequences have such peptides properties that for the complete enzymes according to the invention are characteristic. Such peptides can be produced in large quantities and are particularly suitable for studies on the kinetics of enes enzyme activity, the regulation of enzyme activity, the three dimensions sional structure of the enzymes, the inhibition of enzyme activity  through various chemicals and pharmaceuticals and the binding geometry and binding affinity of different ligands.

Preferably for the recombinant production of the Invention enzymes according to a DNA with the nucleotides from the sequences SEQ ID NO: 1 and 9 used.

As stated above, in plants there is next to the classic Acetate / Mevalonat way an alternative, Mevalonat independent biosynthetic pathway to form isoprenoids, the Deoxy-D-xylulose-phosphate route (DOXP route). It turned out posed that in many parasites, bacteria, viruses and fungi this deoxy-D-xylulose-phosphate pathway also is present.

The invention therefore also includes a method of screening a connection. According to this procedure, becomes a host organ mus containing a recombinant expression vector, wherein the vector according to at least part of the oligonucleotide sequence SEQ ID NO: 1 or SEQ ID NO: 9 or variants or homologues ser, and also a connection suspected of will be an antimicrobial, antiparasitic, antiviral and antifungal effects in humans and animals or a bacterium ricidal, antimicrobial, herbicidal or fungicidal activity Plants. Then the host orga contact with the connection and the effectiveness connection.

Another object of this invention are methods for loading tuning the enzymatic activity of LytB- and YfgB- Protein. This can be determined according to the known techniques the. Here, the change in concentration of the intermediate of the DOXP path, which as substrates or products of the respective Enzymes act through photometric, fluorimetric or chromatographic method determined. Evidence of the conc Change in tration can also be achieved by coupled enzyme assays gene, the proof of one or more additional enzy matic steps taken. The additional enzymes can also  be involved in the DOXP path or experimentally the system be added.

example 1

To check if the lytB gene product is essential for survival Blood stages of the malaria pathogen Plasmodium falciparum necessary an attempt was made to find a "gene disruption" mutant of P. to produce falciparum. In this mutant, gen technical methods a gene that for a selection marker co dated, introduced into the gcpe gene and thereby inacti be fourth. For this purpose, a construct (pPflytBKO) was made, the an expression cassette ver the pyrimethamine resistance averages, contains and from two fragments from the coding Se sequence of the lytB gene is flanked by P. falciparum. This Construct should be by homologous recombination across the flankie sequences are integrated into the gcpe gene.

All PCR amplifications described were performed with thermostabi The Pwo DNA polymerase performed, making the products smooth preserved ends and are suitable for "blunt end" ligations. The sequence of the lytB gene was primed with 5'-ATG TCA GTT ACC ACA TTT TGT TCT TTA AAA AAA ACG G-3 'and 5'-GTG ATT TCA TTT TTC TCT TTC TTT TAT CAT C-3 'and genomic DNA from P. falciparum strain 3D7 amplified as template, with T4- Polynucleotide kinase phosphorylated and in a line with Sma I Arized pUC 19 vector cloned (pUCPflytB). As a selection The marker was the dihydrofolate reductase gene from Toxoplasma gon dii (Tg DHFR-T5) was used, which had been modified so that it imparts resistance to pyrimethamine. The expression of TgDHFR-T5 was not under the control of the 5'- and 3'- translated elements of P. falciparum-calmodulin (Pf CAM) - Gene. This expression cassette was made from the plasmid pTgD- TS.CAMS / 3.KP won, which according to published protocols had been structured (Crabb, B. S. and Cowman, A. F. (1996) Proc. Natl. Acad. Sci. USA, 93, 7289-7294). The Expressionkas sette was amplified with primers 5'- AATCTCTGAGCTTCTTCTTTG-3 'and 5'- GGGGGAGCTCGAACTTAATAAAAAAGAGGAG-3 'with pTgD-TS.CAM5 / 3.KP as Template won. Then the expression cassette  inserted in the insert of pUCPfgcpe. For this purpose, pUCPflytB was included Dsa I opened in the inserts and the overhangs with T4 and Klenow DNA polymerase replenished. The amplified expression cassette te was phosphorylated and inserted via "blunt end" ligation whereby pPflytBKO was obtained.

For transfection by electroporation, the infected erythrocytes (strain 3D7, mainly ring stages, approx. 15% parasitemia) were pelleted in a 10 cm culture dish and in 0.8 ml cytomix (120 mM KCl; 0.15 mM CaCl ₂ ; 2 mM EGTA; 5 mM MgCl ₂ ; 10 mM K ₂ HPO ₄ / KH ₂ PO ₄ ; 25 mM HEPES, pH 7.6), which contained 150 µg plasmid DNA from pPflytBKO. The electroporation was carried out in 4 mm cuvettes at 2.5 kV, 200 ohms and 25 µF. The parasites were then plated out again on culture dishes and incubated. 48 h after the transfection, 400 nM pyrimethamine was added to the culture medium and after a further 48 h the pyrimethamine concentration was reduced to 100 nM. Resistant parasites could be detected microscopically after 22 days. After 6 weeks, the pyrimethamine concentration was increased to 2 µM for a further 3 weeks. The parasites were cloned by limiting the dilution to 96 well cell culture plates and cultivated for 11 days in the absence of pyrimethamine. Then 1 µM pyrimethamine was added again. Culture in the absence of pyrimethamine means that episomal plasmids are lost, and subsequent reselection can only survive parasites that have integrated the plasmid chromosomally.

Parasites grew in only 5 wells since the plasmid appears to be was episomal in most parasites. With these clones was able to trace the expression of the lytB gene by RT-PCR be shown. The plasmid was thus by non-homologous Re combination integrated into the genome and the lytB gene of the para sites not deactivated. Apparently there are parasites with inac activated lytB gene not viable and eat the gene with it tiel. According to recent knowledge, the genus Plasmodium is available phylogenetically lower algae (Fichera, M. E. and Roos, D. S. (1997) Nature, 390, 407-409; Köhler, S, Delwiche, C.F., Denny, P.W., Tilney, L.G., Webster, P., Wilson, R.J.M., Palmer, J.D. and Roos, D.S. (1997) Nature, 275, 1485-1489).  

It can therefore be deduced that the lytB gene obviously also for Plants is essential.

Example 2

To check if the yfgB gene product is essential for survival Blood stages of the malaria pathogen Plasmodium falciparum necessary an attempt was made to find a "gene disruption" mutant of P. to produce falciparum. In this mutant, gen technical methods a gene that for a selection marker co dated, inserted into the yfgB gene and thereby inacti be fourth. For this purpose, a construct (pPfyfgBKO) was made, the an expression cassette ver the pyrimethamine resistance averages, contains and from two fragment from the coding Se sequence of the yfgB gene is flanked by P. falciparum. This Construct should be by homologous recombination across the flankie sequences are integrated into the gcpe gene.

All PCR amplifications described were performed with thermostabi The Pwo DNA polymerase performed, making the products smooth preserved ends and are suitable for "blunt end" ligations. The yfgB sequence was primed with 5'-ATG GAA AAG TCA AAA AGG TAC ATA AGC CTG-3 'and 5'-AGC ATC GTC CAA ACG ATG AAA ATT TTC GTC-3 'and genomic DNA from P. falciparum strain 3D7 amplified as a template, with T4 polynucleotide kinase phospho rylated and into a pUC 19 vector linearized with Sma I cloned (pUCPfyfgB). Dihydrofo was used as the selection marker latreductase gene from Toxoplasma gondii (Tg DHFR-T5) used that had been modified to be resistant to Pyri methamine mediates. The expression of TgDHFR-T5 was un control of the 5 'and 3' untranslated elements of the P. falciparum calmodulin (Pf CAM) gene. These expressions cassette was obtained from the plasmid pTgD-TS.CAM5 / 3.KP, which was constructed according to published protocols (Crabb, B. S. and Cowman, A.F. (1996) Proc. Natl. Acad. Sci. USA, 93, 7289-7294). The expression cassette was amplified with the primers 5'-AATCTCTGAGCTTCTTCTTTG-3 'and 5'- GGGGGAGCTCGAACTTAATAAAAAAGAGGAG-3 'with pTgD-TS.CAMS / 3.KP as Template won. Then the expression cassette inserted in the insert of pUCPfyfgB. For this purpose, pUCPfgcpe was included  Pac I in the insert opened and the overhangs with T4 and Klenow DNA polymerase replenished. The amplified expression cassette te was phosphorylated and inserted via "blunt end" ligation whereby pPfyfgBKO was obtained.

For transfection by electroporation, the infected erythrocytes (strain 3D7, mainly ring stages, approx. 15% parasitemia) were pelleted in a 10 cm culture dish and in 0.8 ml cytomix (120 mM KCl; 0.15 mM CaCl ₂ ; 2 mM EGTA; 5 mM MgCl ₂ ; 10 mM K ₂ HPO ₄ / KH ₂ PO ₄ ; 25 mM HEPES, pH 7.6), which contained 150 µg plasmid DNA from pPfyfgBKO. The electroporation was carried out in 4 mm cuvettes at 2.5 kV, 200 ohms and 25 µF. The parasites were then plated out again on culture dishes and incubated. 48 h after the transfection, 400 nM pyrimethamine was added to the culture medium and after a further 48 h the pyrimethamine concentration was reduced to 100 nM. Resistant parasites could be detected microscopically after 18 days. After 6 weeks, the pyrimethamine concentration was increased to 2 µM for a further 3 weeks. The parasites were cloned by limiting the dilution to 96 well cell culture plates and cultivated for 11 days in the absence of pyrimethamine. Then 1 µM pyrimethamine was added again. The culture in the absence of pyrimethamine causes episomal plasmids to be lost, and only parasites that have integrated the plasmid chromosomally can survive the subsequent reselection. The renewed addition of pyrimethamine was not survived by any parasite clone. This result suggests that parasites with the yfgB gene inactivated are not viable and that the gene is therefore sensitive. According to recent findings, the genus Rias modium is closely related to phylogenetically lower algae (Fichera, ME and Roos, DS (1997) Nature, 390, 407-409; Köhler, S. Delwiche, CF, Denny, PW, Tilney, LG, Webster, P ., Wilson, RJM, Palmer, JD and Roos, DS (1997) Nature, 275, 1485-1489). It can therefore be deduced that the yfgB gene is obviously also essential for plants.

SEQUENCE LOG

Claims (21)

1. DNA sequences for a polypeptide with the in SEQ ID NO: 5 encode amino acid sequence shown for or an analog or derivative of the polypeptide according to SEQ ID NO: 5, wherein one or more amino acids are deleted adds or has been substituted by other amino acids are without the enzymatic effect of the polypeptide we to reduce considerably. 2. DNA sequence according to claim 1 with the Darge in SEQ ID NO: 1 put amino acid sequence. 3. DNA sequences for a polypeptide with the in SEQ ID NO: encode the amino acid sequence shown or for an analog or derivative of the polypeptide according to SEQ ID NO: 4, wherein one or more amino acids are deleted added or substituted by other amino acids are without the enzymatic effect of the polypeptide we to reduce considerably. 4. DNA sequence according to claim 3 with the Darge in SEQ ID NO: 9 put amino acid sequence. 5. DNA sequence according to one of claims 1 to 4, characterized ge indicates that they also have functional regulations signals, in particular promoters, operators, enhancers, ribosomal binding sites. 6. DNA sequence with the following partial sequences

• a) promoter which is active in viruses, eukaryotes and prokaryotes and ensures the formation of an RNA in the intended target tissue or cells,
• b) DNA sequences according to one of claims 1 to 4,
• c) 3'-untranslated sequence which leads to the addition of poly-A residues to the 3 'end of the RNA in viruses, eukaryotes and prokaryotes.

7. Expression vector containing one or more DNA Sequences according to one of claims 1 to 4. 8. Protein, which on 1-deoxy-D-xylulose-5-phosphate Metabolic pathway is involved and a) is encoded by encoded in DNA sequence SEQ ID NO: 1 or 9 or b) is made up of DNA sequences which match the DNA sequences SEQ ID NO: 1 or 9 or fragments of these DNA sequences in the DNA Hybridize the area coding for the mature protein or c) is encoded by DNA sequences without degenera tion of the genetic code with the Se defined in b) sequences would hybridize and for a polypeptide code the corresponding amino acid sequence. 9. A protein according to claim 8, which has the amino acid sequences SEQ ID NO: 5 or 14. 10. Plant cells containing DNA sequences according to one of the Claims 1 to 4. 11. Transformed plant cells and regenerated from them transgenic plants containing DNA sequences according to a of claims 1 to 4. 12. Transgenic viruses, eukaryotes and prokaryotes with isoprene noid expression, characterized in that it is a DNA Sequence according to one of claims 1 to 4 included. 13. Use of a DNA sequence according to one of claims 1 to 4 to determine the enzymatic activity of the LytB- and YfgB protein. 14. Use of a DNA sequence according to one of claims 1 to 4 to change, especially increase the isoprene Noid content in viruses, eukaryotic and prokaryotic Cells.   15. Use of DNA sequences according to one of claims 1 to 4 for the identification of substances containing an inhi have an effect on the LytB and YfgB protein. 16. A method for isolating a protein according to claim 8, characterized in that culture supernatants from parasites or from disrupted parasites via chromatographi and electrophoretic techniques are cleaned up. 17. A method for isolating a protein according to claim 8, characterized in that it is the product of a viral, prokaryotic or eukaryotic expression of a is exogenous DNA. 18. Method for determining the enzymatic activity of the LytB and YfgB protein, characterized in that the Change in concentration of substrates, co-substrates and Products is determined. 19. Process for the production of transgenic viruses, eukaryon ten and prokaryotes with isoprenoid expression, thereby characterized in that a DNA sequence according to claim 4 or 5 in the genome of viruses, eukaryotic and prokaryonti cells with or without the use of a plasmid is transferred and installed. 20. A method of screening a compound, the method comprising:

• a) providing a host cell which contains a recombinant expression vector, the vector having at least part of the oligonucleotide sequence according to SEQ ID NO: 1 or SEQ ID NO: 9 or variants or analogues thereof, and also a compound which is suspected, that it has an antifungal, antibiotic, antiparasitic or antiviral effect on humans and animals,
• b) bringing the microorganism into contact with the connection and
• c) Determination of the antifungal, antibiotic, antipa rasitär or antiviral activity of the compound.

21. A method of screening a compound, the method comprising:

• a) providing a host cell which contains a recombinant expression vector, the vector having at least part of the oligonucleotide sequence according to SEQ ID NO: 1 or SEQ ID NO: 9 or variants or analogues thereof, and also a compound which is suspected, that it has a bactericidal, fungicidal or herbicidal action in plants,
• b) bringing the microorganism into contact with the compound and determining the bactericidal, fungicidal or herbicidal activity of the compound.